Liquid biopsy identification of ERBB2 amplified and HER2 expressing metastatic breast cancer: Comparison and combination of cell and cell-free platforms.
Authors: Giuseppe Di Caro, Ernest Lam, David Bourdon, Tatjana Singer, Kandra Horne, Megan Slade, Rick Wenstrup, Lee S. Schwartzberg
Journal: Journal of Clinical Oncology (2023)

3061 Background: Liquid biopsies are a non-invasive diagnostic approach for detecting circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) that may provide clinically actionable information for treatment decisions for metastatic breast cancer (MBC) patients when a conventional biopsy is otherwise infeasible. Here we report a comprehensive liquid biopsy platform including: 1) quantitative immunofluorescent HER2 and ER protein expression in CTCs (ctcIF), 2) determination of ERBB2 amplification and the number of Large-scale State transitions (LST) by single-cells CTC genomics (ctcDNA) and 3) alterations in plasma ctDNA. Methods: Blood samples from 457 progressing metastatic breast cancer patients and 25 healthy donors (HDs) as controls were collected for cell &amp; cell-free DNA analysis. After plasma isolation, nucleated cells were plated, &amp; slides were bio-banked for Immunofluorescent staining &amp; subsequent imaging. CTCs were identified using Epic Sciences digital imaging &amp; machine learning algorithms, and individual CTCs were sequenced for genomic quantification of LSTs and Copy Number Variants (CNV). Bio-banked plasma was analyzed to detect ctDNA alterations with high clinical relevance (Class IA). Results: Within this cohort of 457 MBC patients, the presence of CTCs, ctcDNA (LST + ) and ctDNA alterations were detected in 98%, 58%, and 42%, respectively, while no CTCs and no ctDNA alterations were detected in the HD cohort, suggesting high specificity. ctcDNA genomics was more sensitive than ctDNA in detecting ERBB2 amp in MBC patients (16%, and 2%, respectively). A mean variant allele frequency (VAF) of &lt; 25%, which is the threshold for detecting a two-fold ERBB2 amplification by ctDNA, was present in 20% and 80% of ERBB2 amp MBC detected by the ctDNA and ctcDNA platform, respectively, suggesting that the ctcDNA platform can identify the majority of ERBB2 amp among patients with a low ctDNA fraction. HER2 exp or ER exp expression by ctcIF were detected in 67% and 67% of MBC patients, respectively. A liquid biopsy classification of HER2 status by combining the three platforms (ctcIF, ctcDNA, and ctDNA) identified that in the tested population, 16% were ERBB2 amp , 52% were HER2 expressing (HER2 exp and ERBB2 nonamp ), and 27% were HER2 neg (HER2 - and ERBB2 nonamp ). To identify MBC patients that may potentially change treatment as a result of the test we identified a small cohort of MBC patients (n=46) with HER2- disease and tissue biopsy IHC=0, 74% of these patients had HER2 exp CTC consistent with HER2 low expression which may aid therapeutic decisions in later line MBC. Conclusions: Here we report a comprehensive liquid biopsy profile combining ctcIF, ctcDNA, and ctDNA platforms with high sensitivity and specificity in determining clinically actionable HER2 and ER biomarker status that may impact therapeutic decision-making in MBC patients.